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United States Department of Agriculture

Agricultural Research Service

Research Project: TRANSGENE MANAGEMENT THROUGH SITE-SPECIFIC RECOMBINATION

Location: Plant Gene Expression Center Albany_CA

Title: Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Psuedomonas syringe type III effector HopZ2

Authors
item LEWIS, JENNIFER
item Wan, J -
item Ford, R -
item Gong, Y -
item Fung, P -
item Wang, P -
item Desveaux, D -
item Gittman, D -

Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 9, 2012
Publication Date: January 20, 2012
Citation: Lewis, J.D., Wan, J., Ford, R., Gong, Y., Fung, P., Wang, P.W., Desveaux, D., Gittman, D.S. 2012. Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Psuedomonas syringe type III effector HopZ2. Biomed Central (BMC) Genomics. 13:8.

Interpretive Summary: Pseudomonas syringae is a bacterial pathogen that infects more than 100 plant species. P. syringae causes disease by injecting type III effector proteins into the plant. The effector proteins target and suppress defense proteins in the plant in order to promote bacterial growth. In order to identify host defense proteins that are targeted by bacterial effector proteins, we developed a high-throughput quantitative protein interaction (yeast two-hybrid) screen. This approach produces quantitative results that can be easily compared among laboratories, and enables high-throughput analysis by eliminating the need to individually sequence DNA. We validate this technique by identifying a plant susceptibility factor (MLO2) that is targeted by a bacterial effector protein. This is a powerful technique to identify host proteins that contribute to many different aspects of plant development.

Technical Abstract: Background: Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system. Results: Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data makes it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence. Conclusions: We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

Last Modified: 9/10/2014